Plasmid Purpose:

This plasmid yields high levels of protein expression from the CMV promoter in mammalian cells but it contains no start codon in the MCS unlike most of our other plasmids. This allows you to insert genes into the downstream restriction sites and use your own genes start codon if required thereby preventing premature translation initiation at the start codon that was within the NcoI site.

Promoter Expression Level:

This plasmid contains the mammalian CMV promoter to drive gene expression. We have tested all of our mammalian promoters in a range of cell types and CMV is consistently the strongest in those we have studied. However there are many reports of the CMV promoter demonstrating silencing by methylation in long-term culture. For this reason we stock a range of other promoters that are compatible with this plasmid and are available on request.

This plasmid contains three alternative transcription terminators for mammalian bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.

Cloning

Cloning in a Gene:

This plasmid has been designed to be compatible with a range of cloning techniques. The multiple cloning site contains a range of standard commonly used restriction sites for cloning. Using these sites genes can be inserted using standard cloning methods with DNA ligase. Other methods such as ligase independent cloning (LIC) Gibson Assembly InFusionHD or Seamless GeneArt can also be used and because all of our plasmids are based on the same backbone the same method can be used for cloning into all of our catalogue vectors.

Multiple cloning site notes:

This multiple cloning site has been modified to remove the start codon within the NcoI restriction site. This was achieved by cleaving pSF-CMV-Amp with KpnI followed by re-circularisation. Removing this site also removes the built in Shine-Dalgarno site and Kozak site from the MCS this will need adding to any gene you clone in to ensure efficient expression.

Within the multiple cloning site there are a few important sites within the MCS. These include the XbaI site and the BsgI and BseRI sites. The XbaI site contains a stop codon. This stop codon is positioned in a specific position in relation to the BsgI and BseRI sites that are immediately downstream. When either BseRI or BsgI cleave the plasmid they produce a TA overhang from the stop codon in the XbaI site that is compatible with all of our peptide tag plasmids cut with the same sites. BseRI and BsgI sites are non-palindromic and cleave a defined number of bases away from their binding site.

IP Status

Intellectual Property Status

This product is part of our SnapFast™ plasmid range, for more information on the intellectual property status of this plasmid and the terms of our licences please click here.